The genus Clostridium encompasses rod-shaped Gram-positive bacteria which are obligate anaerobes capable of producing endospores. Clostridium includes common free-living bacteria as well as important pathogens which include C. botulinum, C. difficile, C. perfringens, C. tetani, and C. histolyticum. The latter is known for producing collagenases which are exotoxins and act as virulence factors, e.g. by facilitating the spread of gas gangrene. Collagenases normally target the connective tissue in muscle cells and other body organs. Owing to the potent hydrolytic activity toward connective tissue, collagenases and other proteinases such as thermolysin are used for tissue dissociation in vitro.
C. histolyticum can be isolated from soil. However, the bacterium is rarely found in wounds. C. histolyticum is not strictly anaerobic and can grow weakly on aerobically incubated media. In culture, proteins and peptides are the main carbon source of C. histolyticum which secretes a variety of enzymes capable of hydrolyzing peptidic bonds.
For technical applications, collagenases from C. histolyticum, i.e. the collagenase of type I and type II, are of particular importance, e.g. for dissociation of organ tissue in vitro. Importantly, collagenase digestion of pancreatic tissue is presently used in the preparation and isolation of human islet cells. However, a number of other different specific cell types have been isolated from attendant connective tissue, including fat cells from adipose tissue, hepatocytes from liver, chondrocytes from cartilage, myocytes from heart, and osteoblasts from bone.
A practical advantage is that C. histolyticum can be cultured in large quantities in simple liquid media, and it regularly produces amounts of proteolytic enzymes which are secreted into the culture medium.
RO 51768 discloses a medium for growing Clostridium histolyticum in order to produce collagenase, whereby the medium contained peptone and paraffin oil.
RU 2180002 discloses a medium for fermentation and purification of collagenase of Clostridium histolyticum comprising casein and soybean oil cake hydrolyzate. Further ingredients were sodium dihydrogen phosphate monohydrate, potassium hydrogen phosphate dihydrate, pyridoxine, riboflavin, thiamine bromide, folic acid, calcium pantothenate, nicotinic acid, lipoic acid, and biotin.
WO 2003/025136 discloses a gelatin-based fermentation medium for the production of cellulose and collagenase from Clostridium collagenovorans. 
WO 2007/089851 discloses media compositions and processes useful for fermentation of C. histolyticum. Proteinaceous constituents of the media included phytone peptone, yeast extract, proteose peptone, tryptone and various vegetable extracts. It was found that a porcine-derived proteose peptone supported the growth of the C. histolyticum strains which under these conditions secreted collagenase enzymes into the culture broth. The culture medium with proteose peptone was optimized in order to reduce the content of the co-secreted clostripain protease.
The culture of C. histolyticum has historically required the use of animal-derived products such as brain heart infusion (e.g. Jozwiak, J., et al., Enzyme and Microbial Technology 39 (2006) 28-31). However, the requirement of proteinacious material of mammalian origin in the media gives rise to concern over possible contamination of the media. In particular, concern that the media may be contaminated with any transmissible spongiform encephalopathy (TSE) causative agent or other infectious and harmful agents, restricts the usefulness of any factors derived from such cultures, especially in therapeutic applications.
Thus, the cultivation media for C. histolyticum of the state of the art have certain disadvantages. In view of this it is an objective of the invention to provide alternative compositions for media to support the growth of C. histolyticum. A particular focus of the invention is the provision of growth media free of mammalian-derived ingredients. Furthermore, the invention aims at providing liquid media are useful for fermentation of C. histolyticum. Additionally, it is an objective of the invention to provide media which support secretion of enzymes with collagenase activity into the culture medium, and preferably in amounts which allow economic purification of the enzymes from culture supernatant at a large scale.
The inventors have surprisingly found, that the combination of fish gelatin and peptones derived from non-mammalian sources solves the above technical problem.